MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

NGUYEN HUU HUNG

RESEARCH ON GENETIC DIVERSITY AND GENETIC

PARAMETERS ACCORDING TO GROWTH TRAITS FOR

TIGER SHRIMP (Penaeus monodon Fabricius, 1798) BREEDING

Major: Aquaculture

Major code: 9620301

SUMMARY OF DOTORAL THESIS

KHANH HOA - 2020

2

The doctoral thesis was completed at Nha Trang University

Supervisor:

1. Dr. Nguyen Van Hao

2. Prof. Dr. Lai Van Hung

Reviewer 1: Dr Nguyen Minh Thanh

Reviewer 2: Dr Tran Thi Thuy Ha

Reviewer 3: Dr Dang Thuy Binh

The thesis is protected at the national evaluation council meeting at Nha Trang

University at the time of day 19 month 9 year 2020

The thesis can be found at: National Library; Library of Nha Trang University

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INTRODUCTION

The demand for seeds for commercial farming of tiger shrimp is about 30

billion/year (Directorate of Fisheries, 2017). However, the majority of broodstock

supplied to hatcheries must be imported from abroad and exploited naturally. In order

to meet the demand for tiger shrimp for the 2018 crop season, 30,000 black tiger shrimp

are needed. The source of broodstock largely depends on natural exploitation. Naturally

caught broodstock from the wild bring many consequences, of which the most important

thing is to spread causative pathogens from the wild into the farming models. In practice,

the current demand for tiger prawn of farmers is fully met by domestic production, but

the quality of shrimp seed is always a problem. Black tiger shrimp are not strictly

controlled on pathogens as well as quality, leading to huge losses for shrimp farmers.

Black tiger shrimp is identified as one of the two main shrimp farming species in our

country, the demand for seeds is increasing in quantity and quality, so the active

development of high quality prawn broodstock through breeding selection in Vietnam

is very necessary.

In Vietnam, studies of tiger shrimp before 2000 focused on biology, reproduction

and seed production. From 2000 to the present, the studies of prawn cultivating have

been done mainly to indoor re-cycle system farming technology. Although there have

been a number of tiger shrimp breeding programs conducted in the world but for reasons

related to technology security, these projects are not published and are not transferred.

From the above analysis, it is necessary to conduct a research program on

breeding selection for high quality seed for this shrimp in Vietnam. Student research

topic "Research on genetic diversity and genetic parameters according to growth traits

for tiger shrimp breeding" is carried out in accordance with Decision No. 1081 / QD-

DHNT of the Rector of Nha Trang University, November 23rd, 2015 under the Doctoral

program, specialized in Aquaculture.

The research topic of PhD student is one of the important contents of the state-

level project "Application of quantitative genetics and molecular genetics to create

initial materials for tiger shrimp breeding according to growth traits" under the Project

on development and application of biotechnology in Agriculture and Fisheries to the

Ministry of Agriculture and Rural Development by 2020 by the Research Institute for

Aquaculture II.

4

Objectives of the thesis:

Identifying genetic diversity and basic genetic parameters as a basis for creating

a high-quality genetic variation of tiger shrimp for initial breeding of tiger shrimp

according to growth traits.

To achieve this goal, the thesis has implemented the following contents:

1. Evaluate of genetic variation of initial tiger shrimp populations by

Microsatellite technique.

2. Evaluate the growth of shrimp lines by hybrid method as a basis for initial

material formation for the breeding program.

3. Estimate some basic genetic parameters on tiger shrimp populations have been

established.

The significance of the thesis:

- Scientific significance:

This programe provide a scientific basis for assessing genetic variation of tiger

shrimp populations to create base breeding populations with high genetic diversity by

evaluating shrimp lines by hybrid hybrids and other Estimated results on some genetic

parameters (evaluation of genetic and environmental interactions, heritability, selective

efficacy).

The results confirmed the scientific basis of the theory and practice of breeding

populations that really contributed to improving the genetic quality of black tiger shrimp

populations in Vietnam. Research results are an important foundation for further

research on this subject.

- Practical significance:

The research results have created a selective product of tiger prawn for the first

generation (G1) with good genetic parameters. This is the original source of materials

for generations to choose from. Shrimp breeding is disease-free and grows faster than

shrimp seed produced from wild broodstock. When commercialized, will provide the

market with fast growth, no pathogen. This helps to solve the two biggest limitations of

the current quality of tiger shrimp as disease and slow growth rate which slow the

industry from development in recent years.

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New points of the thesis:

The results of the thesis are novel in genetic numbers published following a

systematic and complete way of basic genetic parameters of black tiger shrimp in

Vietnam. This is a major breakthrough that opens up opportunities and conditions for

further and longer breeding selections on this subject.

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CHAPTER I. LITERATURE REVIEW

1.1. Achievements of breeding programs for aquaculture species in the country

and around the world.

1.1.1 The breeding programs on aquaculture species

Breeding selection programme initially started on Atlantic salmon (Salmo solar)

from 1971 in Norway (Gjedrem et al. 1991, Gjedrem 2005). Quantitative genetics and

statistic algorithms have been applied in breeding selection on rainbow trout

(Oncorhynchus mykiss) in Norway, Poland and Danmark (Gjedrem 2005), cod fish in

Norway (Kolstad et al. 2006), and American catfish (Dunham and Argue 1998), tilapia

(Eknath et al. 1993), Indian rohu, Pacific oyster, Japanese tiger prawn (Hetzel et al.

2000), Whiteleg shrimp (Argue et al. 2002); (De Donato et al. 2005, De Donato et al.

2008), Crawfish (Jerry et al. 2005). The initial findings showed that the efficency of

selection is high, increasing about 4 – 15% per generation.

In Vietnam, programes for breeding selection in aquaculture have been done on

some common species such as silver carp (hypophthalmichthys molitrix), Vietnam silver

carp (H. harmandi) (An and Thien 1987), common carp (Cyprinus carpio) (Trong

1983), GIFT tilapia (O. niloticus) (Dan and Little 2000), Freshwater prawn (Thanh and

et al., 2009), whiteleg shrimp (Ninh et al. 2017), tiger prawn (Nguyen Van Hao and et

al., 2015).

1.1.2. Geographical diversity of the initial material collection to form the breeding

population

Selective programe on Atlantic salmon (S. salar) collected wild fish from fourty

different rivers in Norway (Gjedrem et al. 1991). GIFT tilapia (Genetically Improved

Farmed Tilapia) (O. niloticus) was formulated from eight populations (Bentsen et al.

1998, Eknath et al. 2007). Red tilapia was collected from seven different population in

all over South America. Foundation populations of tiger prawn from Moana company

was collected wildly from different areas in Asea.

In Vietnam, researches for freshwater prawn (M. rosenbergii), initial materials

was selected from 3 lines from Me Kong, Dong Nai, Malaysia) (Thanh et al. 2009).

Breeding selection for common carp was formed from 3 lines of Vietnam, Hungary and

Indonesia (Ninh et al. 2011). Breeding selection was come from seven imported pounder

7

populations (Tran The Muu, 2015).

1.1.3. The evaluation the initial flow of materials for breeding programs

There are two methods to form an initial population for breeding. The first method

is to collect shrimp from a variety of lines, then to randomly mix, unlimited between lines,

in the first generation. This is the method used to form the Atlantic salmon breeding

population in Norway (Gjedrem et al. 1991). The second method is hybridization between

lines, then select with low intensity in the first generation selection. This method is used

to create populations of zebra tilapia (O. niloticus) under the GIFT program (Bentsen et

al. 1998).

1.1.4 Bio-security and disease free maintenance in shrimp breeding selection

Ensuring biosecurity and disease free are a prerequisite for breeding research and

have been applied to The Hawaii - US Institute of Oceanography Breeding Program (OI)

on white shrimp, the program Research on the cultivation of prawn-free broodstock in

Malaysia, the program of processing prawn from CSIRO - Australia, the tiger prawn

breeding program of Moana Company - Hawaii.

1.1.5. The marker technique for breeding program

Fluorescent color has been applied to mark success on worms (Butt et al. 2009),

eels (Imbert et al. 2007), amphibians (Heemeyer et al. 2007), crustaceans (Zeeh and Wood

2009), fish (Doupé et al. 2003, Woods and Martin-Smith 2004, Astorga et al. 2005, Jensen

et al. 2008), on a number of different shrimp species (Godin et al. 1996), crayfish

(Mazlum 2007), lobster (Uglem et al. 1996, Frisch et al. 2006) and giant freshwater

shrimp (Hung et al. 2012).

1.1.6 The evaluation of genetic and environmental interaction

The evaluation of G × E interaction studies has been published a lot in tilapia

(Uraiwan et al. 1995, Maluwa et al. 2006, Bentsen et al. 2012, Khaw et al. 2012, Trọng

et al. 2013), salmon species (Atlantic salmon, rainbow trout, etc.) (Winkelman and

Peterson 1994, Fishback et al. 2002, Kause et al. 2003, Kolstad et al. 2006, Pierce et al.

2008), carp (Cyprinus carpio) (Ponzoni et al. 2005, Wang et al. 2007, Ponzoni et al.

2008), bass family (Lates calcarifer) (Saillant et al. 2006, Dupont-Nivet et al. 2008,

Domingos et al. 2013), oysters (Pinctada maxima) (Kvingedal et al. 2007, Swan et al.

2007).

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1.1.7. Heritability and genetic correlation

Heritability for different traits in shrimp has been published by the authors: Wong

and McAndrew 1990, Benzie et al. 1997, Hetzel et al. 2000, Argue et al. 2002, Goyard et

al. 2002, Gitterle et al. 2005a, Gitterle et al. 2005b.

1.1.8. The selective effect

Selective effects on aquatic subjects are usually quite high, up to 10 to 20% per

generation as in Atlantic salmon (Salmo salar) (Gjedrem 2000, Quinton et al. 2005), coho

salmon (Oncorhynchus kisutch) (Hershberger et al. 1990, Neira et al. 2004), American

catfish (Ictalurus punctatus) (Dunham 2007), tilapia (Oreochromis niloticus) (Bentsen et

al. 1998, Ponzoni et al. 2005, Eknath et al. 2007, Trọng et al. 2013), Indian carp (Labeo

rohita) (Mahapatra et al. 2006) and catfish (Pangasianodon hypophthalmus) (Nguyễn

Văn Sáng et al. 2012). Unlike fish studies, there are very few crustacean breeding

programs and mainly focus on some marine shrimp species (Hetzel et al. 2000, Argue et

al. 2002, Goyard et al. 2002, Preston et al. 2004, De Donato et al. 2005, Gitterle et al.

2005, Gitterle et al. 2006), freshwater crayfish (Jones and Ruscoe 2000, Jerry et al. 2005),

giant freshwater shrimp (Macrobrachium rosenbergii) (Luan et al. 2012, Hung et al.

2014, Hung and Nguyen 2014, Luan et al. 2014, Luan et al. 2015).

1.2. The domestication and completion of closed life cycle of objects selected in

artificial conditions in the country and in the world

Worldwide, shrimp farming began early in the mid-1970s, including whiteleg

shrimp (Liptopenaeus vannamei) and Western blue shrimp (Liptopenaeus stylirostris)

(Cuzon et al. 2004), tiger shrimp (Aquacop 1977, Primavera and Gabasa Jr 1981,

Primavera 1998, Subramaniam et al. 2006). In Vietnam, although many domestic research

programs on broodstock have been published, closing the life cycle of black tiger shrimp

from eggs to broodstock and creating generations of home-grown shrimp in domestic

conditions. For the first time successfully and initially small-scale commercialization, it

is the program "Close up the life cycle and produce disease-free black tiger shrimp and

tiger shrimp" (Hoa et al. 2009).

1.3. Microsatellite techniques in assessing genetic variation of breeding materials

in aquaculture

Today, molecular markers, mainly AFLP and microsatellite, have been widely

9

used to evaluate genetic diversity between natural populations and domesticated pants for

some economically valuable aquatic species such as salmon (Skaala et al. 2004), common

carp (Kohlmann et al. 2005), tilapia (Romana-Eguia et al. 2004); (Xu and Thornalley

2001, Cruz et al. 2004, Dixon et al. 2008).

1.4. Application of modern methods (MAS – Marker Assisted Selection; GWS –

Genome Wide Selection) in breeding program.

Breeding based on molecular markers linked to a trait of interest called marker-

assisted selection (MAS) is a process that uses molecular markers to indirectly select

one or more genetic important traits, this is a method widely supported by the world

because this technique shortens the breeding time and can get results after three selective

generations (Tanksley and Nelson, 1996). Breeding genetic selection based on whole-

genome has been extensively researched and developed for animal husbandry (Solberg

et al., 2008. In aquaculture, a recent review of MAS in fish breeding, Sonesson (2003)

reported that MAS will be valuable for traits that are difficult to record and observe such

as disease resistance, fillet quality, feed efficiency and maturity.

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CHAPTER II. RESEARCH METHOD

2.1. Object, time, place of study and research scope

2.1.1. Research subjects

The research object is black tiger shrimp (Penaeus monodon Fabricius, 1798).

2.1.2. Research time

The duration of the study is from 2013 to 2018, in which the time for data analysis is

from 2012 to 2015.

2.1.3. Research location

- National Center for Seafood Breeding - Institute of Aquaculture Research II.

Implementing the main contents of the thesis.

- National Center for Central Seafood Breeding - Research Institute for

Aquaculture III. Implementation of the evaluation of genetic-environmental interaction.

- Bac Lieu Experimental Research Centre - Aquaculture Research Institute II.

Implementation of the evaluation of genotype-environment interaction (G x E).

- Dang Lam private enterprise (Vung Tau). Implementation of the evaluation of

genotype-environment interaction (G x E).

- Southern Center for Fisheries Environmental and Disease Monitoring -

Research Institute for Aquaculture II. Implement content of screening tests of common

viral pathogens.

2.1.4. The research scope

Research is limited to sources of material collected from the ocean through

suppliers. The studies were conducted in the South Central region and Mekong Delta.

The study of genetic diversity limited to the use of microsatelite tools and genetic

parameters is estimated for two generations G0 and G1.

2.2. Research materials

The research material is broodstock of tiger shrimp collected from Thailand,

Singapore and Vietnam with a total of 460 females and 376 male shrimps.

2.2.1. Research materials for genetic variation evaluation by microsatellite

The pool pattern was randomly collected from four broodstock broodstock with

a total of 137 samples analyzed.

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The primers used for the study was from Sigma, a total of 15 microsattlite

primers were used to evaluate the genetic diversity of the four original tiger shrimp.

2.2.2. The research material was used to evaluate the flow by mixed hybridization and

estimate some basic genetic parameters

- A total of 69 shrimp families from 16 hybrids created G0 generation with the

number of 200 individuals/family, the average size of 2g generated from the original

four shrimp materials of different origins.

- A total of 76 families from first generation (G1) with 7,412 shrimp at the average

size of 2 - 3g.

2.3. Research Methods

2.3.1. Method of assessing genetic variation by microsatellite

Research methods include steps: sample preparation, DNA extraction,

microsatellite amplification by PCR technique, Agarose gel electrophoresis, allen

analysis.

2.3.2. Method of evaluating the flow by mixed hybrid method

2.3.2.1. Maturation methods, broodstock reproduction, larval rearing

The methods of use includes: isolation culturing, broodstock maturation, sperm

insertation, maternal eye cutting, maturation shrimp inspection, reproduction, egg

collection, egg washing and incubation, larval rearing from Nauplius to PL15

postlarvae, nursing from PL15 post-larval stage to mark size.

2.3.2.2 Growth method of shrimp families of generations G0 and G1

- The G0 generation cultured in four locations, including: Khanh Hoa province, Bac Lieu

province, Vung Tau province and biosecurity tanks.

- The G1 generartion cultured in two locations, including: Khanh Hoa province and

biosecurity tanks.

The process of growth adopts the published process and is currently being

applied in localities.

2.3.2.3. Individual marking method

Fluorescent colors (VIE) is used to mark shrimp individuals according to the

manufacturer's instructions (Northwest Marine Technology Company).

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2.4. Experimental layout

2.4.1. Experimental arrangement of selected primers used to evaluate genetic

variation by microsatelite

Surveying 29 primer pairs on 28 random shrimp DNA samples to select 15 primer

pairs using genetic diversity analysis of four primary broodstock.

2.4.2. Arrangement of full hybrid combinations of 4 shrimp populations

Families of G0 generation were created from the combination of all hybrids of 4

shrimp groups (4 x 4 = 16 hybrids) including 4 inbred hybrids (AA, TT, NN and GG)

and 12 cross-bred hybrids (AT, AN, AG, TA, TN, TG, NA, NT, NG, GA, GT, GN).

2.4.3. Hybrid layout of generation G0 to formulate G1 generation

Generation G1 families are bred based on Index value and traceability of pedigree

to avoid inbreeding and the presence of three Index groups with different ratios.

2.4.4. Growth arrangement to evaluate genetic and environmental interaction

Growth culture evaluated genetic and environmental interactions conducted in

earthen ponds in South Central (Khanh Hoa), Southwest (Bac Lieu) and Dong Nam Bo

(Vung Tau) and biosecurity (Trung National Center for Southern Breeding Seafood)

2.5. Methods of data analysis and processing

2.5.1. Methods of analyzing and processing data evaluate genetic diversity

Using GenAlEx software (Peakall and Smouse 2006) Genepop 4.2 to calculate

allele frequencies, observed heterozygotes (H0), expected heterozygotes (He), and

genetic differences (Fst), inbreeding coefficient (FIS), test deviation from the Hardy-

Weinberg Equilibrium.

2.5.2. Methods of analyzing and processing data for assessing growth of different

shrimp lines by mixed hybrid method

Before communal stocking, each families was randomly scaled 30 individuals

to record body weight when starting to place into biosafety tanks and ponds. After

the culture period, about 80 days, all the shrimp was harvested in the tanks and ponds.

Collected data include body weight (g, error of 0.1g), gender (male/female), culture

tank and tagging signals (to retrieve families).

- Least square mean (LSM) estimate

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The linear model selected after screening all fixed and covariance effects was

formulated:

Weightijkl = µ + hybridi + sexj + age + tagging weight

+tankk + (hybrid tank)l + residueijkl

2.5.3. Methods of data collection and statistic analysis for basic genetic descriptic

parameters

Descriptive statistics was affected by sex (different weight between male and

female) and broodstock (male or female broodstock) being analysed by software R 3.2.1

(Core 2015). The variance components are estimated by ASReml 3.0 (Gilmour et al.

2009).

- Genetic and environment interaction for two culture environment

The genetic correlation of the trait of harvest weight between two culture

environments is estimated by the formula

𝒓𝒈 =𝝈𝟏𝟐

√𝝈𝟏𝟐×√𝝈𝟐

𝟐,

Where 𝝈𝟏𝟐 is covariance of cumulative genetic effects của of harvest weight between

two environments, 𝝈𝟏𝟐 and 𝝈𝟐

𝟐 are variance of cumulative genetic effects of harvest

weight from environment 1 and 2 (Falconer and Mackay 1996).

- Methods of estimating heritability

Heritabilities for weight traits was estimated as formula:

And common invironmental effects

.

Heritabilities for survival rate (alive = 1; dead = 0) were estimated as formula.

ℎ2 = 𝜎𝐴

2

𝜎𝐴2 + 𝜎𝐸

2 ×𝜋2

3

with 𝜎𝐸2 was valued as 1 (Gilmour et al. 2009).

- Non-consecutive selection efficiency: 𝑅1 = 𝑖 × ℎ2 × 𝜎𝑃

- Family selection efficiency: 𝑅2 = 𝑖 × 𝑟 × 𝜎𝑃

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CHAPTER III. RESEARCH RESULTS AND DISCUSSION

3.1. Evaluation of genetic variation in black tiger shrimp by microsatellite markers

3.1.1. Results of microsatellite selection for genetic variation

Based on the survey results, 29 microsatellites with good amplification products

were selected. There are 29 pairs of primers being use to run polymorphic analysis on

28 samples of random shrimp DNA. And then, PCR performance and the number of

alleles of each pair of primers to screen were evaluated and then the microsatellite for

best performance were selected. The results of a total of 15 microsatellite markers were

used to analyze and evaluate the genetic variation of the four shrimp populations as

Indian, Pacific, Gia Hoa and Domestic: W2, W3, W4, W9, W10, P1, P2, P4, P5, P7, L1,

L3, L4, N1, N2.

3.1.2. Genetic diversity of 15 microsatellite on original materials of four groups of

black tiger shrimp broodstock

Results of the survey of genetic diversity of 15 microsatellite species in the four

groups of tiger prawn in the beginning showed that only three microsatellite species (P2,

P4 and P5) are distributed in balance with HWE, the remaining microsatellite deviated

from HWE. This is compatible with observed heterozygous results in the study from

0.176 to 0.557, an average of 0.407 lower than the expected heterozygous (0.490 -

0.738), an average of 0.633. The inbreeding value (FIS) is relatively high, averaging

0.357 0.19. A FIS-value of over 0.125 may indicate a state of genetic degradation due

to inbreeding in the population. The above parameters show some limitations on the

general genetic diversity of four broodstock broodstock used as initial materials.

3.1.3. Genetic diversity of four populations of original tiger shrimp

Survey results of genetic diversity within each population showed that the

observed heterozygous genotype index (Ho) in this study has an average value of 0.41

lower than all of the microsatellite coefficients compared to the coefficient. Expected

heterozygous (He) is 0.63. Among the four analytical sample groups, the average PIC

(polymorphic information) of Gia Hoa population is the lowest of 0.515; Three shrimp

populations are equivalent to 0.554. Microsatellites analyzed on domestic shrimp

population distributed in accordance with Hardy-Weinberg equilibrium. The inbreeding

value of FIS for domestic shrimp is the lowest (0.316) compared to the three groups of

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shrimp originating from the Indian Ocean, Pacific and Gia Hoa (0.374; 0.398; 0.338

respectively). The genetic diversity values of the three populations of Indian Ocean,

Pacific and domestic shrimp are relatively equal and high, equivalent to 0.645. The

cultured shrimp has the lowest genetic diversity index of 0.599 and has large variation

in the population (0.398 - 0.710). There are significant genetic differences with the

remaining shrimp populations.

3.2. Evaluation of growth of black tiger shrimp populations

3.2.1. Disease screening on the original broodstock stocks

Results of screening with different PCR techniques for 4 common dangerous

viruses showed that the rate of disease-free shrimp was 48.0% for female shrimp and

54.5% for male shrimp.

3.2.2. Families production and hybrid families

The results of hybridation of 4 shrimp groups have created 69 families (58 full-

sib families and 11 half-sib families) out of 16 hybrid combinations.

The genetic contribution ratio of the Inland group (N) is the highest and the

lowest is the Gia Hoa group (28.7% and 20.4% respectively), the results are shown in

Table 3.1. In theory, the rate of contribution of genetic material for each herd of balanced

shrimp, 25%, is the best.

Table 3.1. Contribition ratio of original shrimp population materials G0

Shrimp

populations

Indian (A) Gia Hoa (G) Domestic (N) Pacific (T)

Sex Female Male Female Male Female Male Female Male

Number 19 10 6 16 17 14 13 13

Contribution

ratio in each

hybrid (%)

34.5 18.9 10.9 30.2 30.9 26.4 23.6 24.5

Total (Male

and female)

29

(26.9%)

22

(20.4%)

31

(28.7%)

26

(24.1%)

16

3.2.3. Rearing result of generation G0 families

3.2.3.1 Rearing from nauplius to 15th postlarvae (PL15)

Rearing result from Nauplius to 15th postlarvae was presented on Table 3.2:

Table 3.2. Rearing result from nauplius to 15th postlarvae

No Hybrids Number of families Survival rate (%)

1 T × T 6 63.3 ± 6.3a

2 T × N 4 57 ± 10.9a

3 T × G 4 59 ± 6.6a

4 T × A 4 60.5 ± 13.9a

5 N × N 6 51 ± 4.3a

6 N × G 6 49 ± 13.3a

7 N × T 5 55.6 ± 8.3a

8 N × A 4 52.5 ± 10.3a

9 G × G 1 50a

10 G × N 2 60 ± 8.49a

11 G × T 2 49 ± 1.41a

12 G × A 2 44 ± 2.83b

13 A × A 7 49.1 ± 23.8a

14 A × T 6 46.7 ± 22.97a

15 A × N 5 60 ± 3.16a

16 A × G 5 54.4 ± 5.6a

Values in the same column with different letter symbols are significantly different

(P<0,05). A: Indian shrimp; T: Pacific shrimp; G: Gia hoa shrimp; N: Domestic shrimp.

3.2.3.2. Shrimp development from 15th postlarvae (PL15) to tagging size

Shrimp after 30 days of rearing from PL15, the average of shrimp families

achieved weight and length of 0.4 g and 2.8 cm respectively. 60-day-old shrimp, the

average weight reached 2.0 ± 0.5 g and length 4.5 ± 0.4 cm.

3.2.4. Growth of farmed shrimp in biosafety tanks

3.2.4.1. Descriptive statistics and affecting factors on G0 shrimp growth

After being marked and stocked for 80 days in a bio-secure tank, body weight

data was measured from 1,803 individuals originating from a full 69 families with an

average weight of 26.1g. The proportion of female shrimp (52.6%) is higher than that

of male shrimp (47.4%). Female shrimp with higher harvest weight (P <0.05) than male

shrimp.

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Table 3.3: Descriptive statistics for shrimp body weight traits

Shrimp group Number of

individuals (N)

Mean body

weight (g)

Variable coefficient

(CV - %)

Whole populations 1803 26.1 ± 0.14 22.2

Female 949 27.8 ± 0.19 a 21.4

Male 854 24.2 ± 0.17 b

20.5

Determining the fixed and covariate elements in the linear model, the result has

5 single elements and a two-way correlation has a significant effect.

3.2.4.2. Shrimp growth among hybrid combinations

The results of the growth comparison between inbred hybrids and cross-bred

hybrids indicate quite significant differences. Other hybrids give higher growth results.

Table 3.4. Results of analysis of growth data of shrimp in hybrid groups

In this study, the impact on the growth of offspring is due to the origin of

Order Hybrids Number of

families

Number of shrimps

(n)

Body weight (g)

Survival rate (%)

1 T × G 4 123 31,6e ± 0,43 87,85c ± 1,42

2 G × G 1 54 30,1de ± 0,77 77,14bc ± 8,08

3 G × N 2 103 28,4cde ± 0,46 85,71bc ± 24,74

4 N × G 6 157 27,5bcd ± 0,41 74,76bc ± 14,27

5 G × A 2 55 27,1abcd ± 0,66 78,57bc ± 22,22

6 A × G 5 139 26,9abcd ± 0,40 78,28bc ± 20,36

7 G × T 2 46 26,7abcd ± 0,77 43,80a ± 8,08

8 N × N 6 147 25,4abc ± 0,41 70,00abc ± 16,92

9 T × N 4 88 25,2abc ± 0,54 62,85abc ± 14,19

10 A × T 6 155 25,0abc ± 0,40 88,57c ± 5,34

11 A × A 7 172 24,9abc ± 0,37 81,90bc ± 24,08

12 T × A 4 110 24,8abc ± 0,45 78,57bc ± 9,75

13 N × T 5 117 24,8abc ± 0,43 66,85abc ± 9,16

14 T × T 6 122 24,6abc ± 0,50 58,09ab ± 18,57

15 A × N 5 139 23,9ab ± 0,42 75,42bc ± 24,71

16 N × A 4 89 23,3a ± 0,51 63,57abc ± 14,63

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broodstock. The influence of broodstock and or prawn factors is unclear.

3.3. Estimation of some basic genetic parameters

3.3.1. Evaluation of genetic and environmental interaction (G × E)

3.3.1.1. Descriptive statistics and influencing factors

Table 3.5. Breeding results grow in different environments

Culturing

location

Factors Shrimp

number (N)

Mean Variable

coefficient

(CV-%)

Biosecure tanks Culturing duration (80 days)

Population weight (g) 1816 26.1 ± 0.14(b) 22.1

Female weight (g) 957 27.8 ± 0.19(a) 21.4

Male weight (g) 859 24.2 ± 0.17(b) 20.5

Khanh Hoa Culturing duration (94 days)

Population weight (g) 4316 29.3 ± 0.08(c) 17.4

Female weight (g) 2229 30.8 ± 0.11(a) 17.0

Male weight (g) 2087 27.8 ± 0.10(b) 16.0

Bac lieu Culturing duration (95 days)

Population weight (g) 4979 25.0 ± 0.08(a) 23.2

Female weight (g) 2531 26.2 ± 0.12(a) 23.2

Male weight (g) 2448 23.8 ± 0.10(b) 21.9

Vung Tau Culturing duration (100 days)

Population weight (g) 3458 26.1 ± 0.13(b) 28.7

Female weight (g) 1697 27.4 ± 0.20(a) 29.5

Male weight (g) 1761 24.8 ± 0.16(b) 26.7

Values for comparing mean weights between female and male at same location

presented by different letters showed significant difference (P<0,05).

19

Determining the fixed and covariate elements in the linear model, the result has

5 single elements and a two-way correlation has a significant effect and is included in

the mathematical model.

3.3.1.2. Correlation between genotype and environment (G × E)

The analytical results show that G × E correlation is positively correlated (> 0)

and lies from low to high from 0.29 to 0.85. There is a high genetic correlation between

culture in biosecurity tank and Khanh Hoa culture site (0.70), Khanh Hoa and Bac Lieu

(0.74); The correlation with the two sites of Bac Lieu and Vung Tau is 0.42 and 0.29

respectively.

Table 3.6. Correlation of genotype (rg) of body mass traits in different culture

environments for G0 vaf G1

Culturing

locations

G0 generation G1 generation

Biosecurity

tanks

Khanh Hoa Bac Lieu Vung Tau Khanh Hoa

Biosecurity tanks 0,70 ± 0,09 0,42 ± 0,13 0,29 ± 0,15 0,75 ± 0,09

Khanh Hoa 0,74 ± 0,08 0,51 ± 0,12

Bac Lieu 0,85 ± 0,05

3.3.2. Heritability

The results of formulating families, culturing and evaluating the growth of G1

generation of shrimp families are presented in Table 3.7 and 3.8.

Table 3.7. The number of full-sib and half-sib families in G1 generation

Family

type

Total

families

Number of

shrimp

Male

×

Female

Full-sibs

families

Half-sibs

families

Half-sibs

families 50 5155 1 × 2 6 12

1 × 3 3 9

1 × 4 3 12

1 × 5 2 10

1 × 7 1 7

Full-sibs

families 26 2257 1 × 1

Total 76 7412

20

Table 3.8. Quantity and weight of shrimps at harvesting time in two ponds in Khanh

Hoa and in biosecure tanks

Environm

ents

Batch Sex Quantity Weight at harvest time (g)

Mean

weight

(g/shri

mp)

Stand

ard

devia

tion

(SD)

Varia

ble

coeffi

cient

(%)

Min

(g/s

hrim

p)

Max(g/s

hrimp)

Khanh

Hoa

(pond)

1 Female 303 23.26 7.90 33.97 5.77 53.59

Male 256 20.94 7.10 33.93 7.52 50.34

2 Female 938 20.99 6.24 29.72 5.72 42.65

Male 794 19.51 5.13 26.28 5.88 37.33

3 Female 559 29.81 7.66 25.70 9.03 58.71

Male 451 26.53 7.18 27.08 8.04 54.41

The total number of shrimp harvested at Khanh Hoa: 3301 individuals

Biosecure

tanks

1 Female 672 22.70 7.17 31.82 6.25 49.60

Male 646 20.53 6.24 30.41 6.55 42.55

2 Female 1009 24.26 8.24 33.96 7.06 56.40

Male 914 21.64 6.50 30.06 7.16 42.83

3 Female 461 24.13 7.29 30.20 7.20 46.60

Male 409 21.95 6.78 30.88 5.90 42.60

The total number of shrimp harvested at biosecure tanks: 4.111 individuals

The total harvested shrimp was 7412 of which 3301 were from pond culture and

4111 from tank culture. The average weight of cultured shrimp in ponds ranged from

19.51 ± 5.13 to 29.81 ± 7.66 g/shrimp, and shrimp in tanks with weight from 20.53 ±

6.24 to 24.26 ± 8.24 g/shrimp. The coefficient of variation in cultured shrimp weight is

high, from 25.70 to 33.97% for shrimp cultured in ponds and 30.06 to 33.96 g/shrimp

for tank shrimp.

- Estimating heritability

For the G1 shrimp weight trait at harvest, the estimate of the variance components

(𝜎𝐴2, 𝜎𝐶

2 và 𝜎𝑃2), the heritability factor (h2) and the effect of common environmental

effect (c2) for the two culture environments in Khanh Hoa and the biosecure tanks are

shown in Table 3.9.

21

Table 3.9. The variance components (𝜎𝐴2, 𝜎𝐶

2, 𝜎𝑇𝑁2 , 𝜎𝐸

2,và 𝜎𝑃2) and heritabilities (h2)

and common environmental effects (c2) on shrimp weight of G1 generation (±SE)

Environments 𝜎𝐴2 𝜎𝐶

2 𝜎𝑇𝑁2 𝜎𝐸

2 𝜎𝑃2 h2 c2

Khanh Hoa 37.47 12.11 4.68 8.47 62.73 0.60

0.17

0.19±

0.09

Biosecure

tanks

35.54 10.64 2.76 14.53 63.47 0.56

0.15

0.17±

0.09

Heritability for weight traits is estimated at a high level, shrimp in ponds is 0.60

± 0.17 and in tanks is 0.56 ± 0.15. The impact of the environment was also large, shrimp

ponds in Khanh Hoa were 0.19 ± 0.09 and cultured in the biosecure tanks was 0.17 ±

0.09. The genetic correlation of shrimp weight when harvested between two culture

ponds and tanks was estimated at 0.75 ± 0.09, indicating a moderate environmental

interaction.

For the trait of survival and estimation of heritability for the two culture

environments in Khanh Hoa and biosecure tanks is presented in Table 3.10. Heritability

was estimated to be quite good and comparable between the two environments, for

shrimp farming in Khanh Hoa was 0.18 ± 0.02 and in biosecure tank was 0.19 ± 0.02.

For both environments, the genetic correlation between survival and harvest weight is

positive and relatively low, with 0.40 ± 0.08 in Khanh Hoa and 0.29 ± 0.08 in biosecure

tanks.

Table 3.10. Heritability (h2) of survival and genetic correlation (rg) between

individual survival rate and shrimp harvest weight in Khanh Hoa and biosecure

tanks

Traits h2

rg

Weight in

Khanh Hoa

Weight in

Biosecure tanks

Traits in Khanh Hoa (%) 0.18 ± 0.02 0.40 ± 0.08

Traits in biosecure tanks (%) 0.19 ± 0.02 0.29 ± 0.08

The correlation between survival rate and estimated breeding value (EBV) of

survival rate in each environment and between Khanh Hoa and biosecure tanks is shown

in Table 3.11. The correlation between survival rate and EBV of survival rate is high,

for Khanh Hoa is 0.80 and for biosecure tanks is 0.89. Between the two environments,

22

the correlation between survival rate and EBV survival rate is a positive correlation,

ranging from 0.37 to 0.55.

Table 3.11. Correlation between survival rate and estimated breeding value (EBV) of

survival rate in Khanh Hoa and biosecure tanks

Traits Survival rate in

biosecure tanks

EBV survival in

Khánh Hòa

EBV survival

in biosecure

tanks

Survival rate in

Khánh Hòa (%)

0.46 0.80 0.37

Survival rate in

biosecure tanks (%)

0.43 0.89

EBV survival in

Khánh Hòa

0.55

3.3.3. Selection efficiency after a generation of growth traits

3.3.3.1. Selection efficiency

For population G0, the efficiency in non-consecutive selection (R1) is 0.9g,

corresponding to 3.3%. For G1 population, selective efficiency R1 is 4.3 g (19.2%).

When selecting families, R2 is similar to that of R1, giving G0 of 0.9 g (3.3%) and giving

G1 of 4.1 g (18.1%).

Table 3.12. Selection rate, selective intensity, heritability, standard deviation of the

weight trait and selection efficiency of two populations of tiger shrimp G0 and G1

Generation R p

(%)

i h2 𝝈𝑷 R

(g)

Weight

(g)

Gain

(%)

G0 R1 80.4 0.35 0.43 5.7 0.9 26.1 3.3

R2 80.4 0.35 0.43 5.7 0.9 26.1 3.3

G1 R1 34.1 1.06 0.56 7.3 4.3 22.6 19.2

R2 34.1 1.06 0.53 7.3 4.1 22.6 18.1

p = selection rate, i = selection intensity (Falconer and Mackay 1996), h2 = heritability,

r = selection efficiency = √1

2ℎ2, 𝜎𝑃= standard deviation of harvest weight, R1 = non-

consecutive selection efficiency and R2 = family selection efficiency.

23

CHAPTER IV. CONCLUSION AND RECOMMENDATION

4.1. Conclusion

1. The some limitations on the general genetic diversity of four broodstock

sources used as initial materials. The results of heterozygous observations averaged

0.407 (0.176 to 0.557) is lower than expected heterozygosity (0.490 - 0.738), an average

of 0.633. The inbreeding index (FIS) is relatively high, averaging 0.357 0.19.

2. Within the scope of the study, the genetic material of the Domestic population

is relatively good. Domestic shrimp population have distribution in compliance with

HWE equilibrium compared to the remaining shrimp populations. The FIS inbreeding

value of the Domestic group was the lowest (0.316) compared to the three populations

of Indian, Pacific, and Gia Hoa (0.374; 0.398 and 0.338). The genetic diversity values

of the three populations of Indian, Pacific, and Inland shrimp are relatively equal and

high, equivalent to 0.645.

3. The average PIC index (polymorphic information) of the cultured shrimp is

the lowest of 0.515 compared to the remaining shrimp (~ 0.554). The cultured shrimp

has the lowest genetic diversity index of 0.599 and has large variation in the population

(0.398 - 0.710). There is a genetic difference of the cultured shrimps with three

remaining populations (P ≤ 0.001). The difference between pairs of shrimp (Indian,

Pacific), (Indian Ocean, Inland) and (Pacific, Inland) is not significant.

4. High variability in phenotype of body mass traits in both male and female

shrimp (about 21.3%) allows the prediction of tiger shrimp populations in this study to

have potential to improve quality in further breeding selection.

5. The impact on growth of offspring is due to the origin of broodstock, the

influence of broodstock and / or prawn factor is unclear. The results of the growth

comparison between inbred hybrids and other crossbred hybrids show quite significant

differences, in which other hybrid hybrids produce higher growth results. In inbred

hybrids, the hybrid combination of Gia Hoa shrimp (G x G) produces the best growth

results. For hybrids of wild-caught shrimp populations, the domestic herd cross-bred (N

x N) represents the dominant phenotype of growth traits compared to the remaining two

groups of shrimp (A x A and T x T).

24

6. Most of the hybrids that yield the best growth in phenotype are inbred

crosses. Out of the six crosses that yield the best results (except for in-line GG), all are

of the Gia Hoa (G) line with the other three. Off-line crosses between wild-caught

shrimp (eg, NA, NT, TA, etc.) often show lower growth.

7. All genetic correlations of stem body traits harvested in different culture

media were positive, inconsistent and range from low to high (rg from 0.29 to 0.85). (>

0), which allows mild GxE interactions to be predicted.

8. Genetic correlation (rg) of shrimp weight when harvested between two culture

environments in Khanh Hoa and biosecure tanks (G1 generation) is estimated to be 0.75

0.09. Besides, the interaction of G x E (generation G0) is low (rg = 0.42) of weight

trait between biosecure tanks and ponds in Bac Lieu, where is the main farming region

requiring to maintain and further enhance the genetic diversity of this material source

in order to better adapt to different types of environments and farming models.

9. Genetic correlation between trait of survival rate and harvest weight ranged

from 0.29 ± 0.08 (biosecure tanks) to 0.40 ± 0.08 (Khanh Hoa), indicating that selection

was improved. Growth will not negatively affect the survival rate of farmed shrimp. The

genetic correlation between EBV survival rate and survival rate in each environment

was high (0.80 for Khanh Hoa and 0.89 for biosecure tanks). Between the two

environments, the correlation between survival rate and EBV survival rate is a positive

and relatively low correlation ranging from 0.37 to 0.55 due to the nature of the two

culture environments and the care regime is very different.

10. The heritability of shrimp weight is estimated at a high level, for shrimp

farming in Khanh Hoa is 0.60 ± 0.17 and for shrimp in the biosafety tank is 0.56 ± 0.15.

The effect of the environment was also quite good, for Khanh Hoa was 0.19 ± 0.09 and

for the biosecure tanks was 0.17 ± 0.09. For the individual survival rate, the estimate of

heritability for farmed shrimp in Khanh Hoa was 0.18 ± 0.02 and the biosecure tanks

was 0.19 ± 0.02.

11. The selective effect for population G0 in the case of non-consecutive selection

(R1) is 0.9 g, corresponding to 3.3%. For G1 population, R1 is 4.3 g (19.2%). When

selecting families, R2 is similar to that of R1, giving G0 of 0.9 g (3.3%) and giving G1 of

4.1 g (18.1%).

25

4.2. Idea suggestion

1. Results of evaluation of genetic diversity in the whole population and each of

broodstock as initial materials for the breeding process suggest that it is necessary to

collect and supplement the diverse shrimp stocks. especially paying attention to

populations of naturally derived shrimp.

2. Continuing researches to reduce the impact of environmental factors (c2) such

as well implementing the complete hybrid structure (hierarchical hybrid structure ) is

needed, reducing family production time, shrimp tagging size (i.e. reducing the duration

of a separate family nursing time). The procedure for techniques of care and nutrition in

the pre- and mature stages is complete, improving the rearing and rearing system in

different farming environments to contribute to increasing the accuracy of the

heritability estimation.

3. The use molecular genetics will be continued to support genetic numbers in seed

selection to accelerate selective efficiency.